Allostery remains an important concept in molecular biophysics that is used extensively to explain the functionality in many multi-subunit protein systems. Hemoglobin (Hb) has been and continues to be the prototypical allosteric model protein. Although many features of Hb reactivity are explainable in terms of the two-state MWC model for allostery, several recent studies indicate substantial deviations from this ideal behavior. The challenge in addressing the extent and basis for these potentially important deviations arises from both the need to isolate ligand binding intermediates and the need for site specific probes that are suitable for testing stereochemical models. In this project several site-specific optical probes are to be used to probe both local tertiary conformations and global quaternary states. CW and time-resolved visible resonance Raman and near IR absorption will probe the heme and its environment, UV resonance Raman will probe the alpha1beta2 interface and a new fluorescence lifetime technique will be used to probe the R-T sensitive 2,3-DPG binding site. Geminate recombination in photo-dissociated COHbs is to be used as a probe of heme reactivity. These tools will be used in conjunction with a newly developed sol-gel encapsulation technique which allows for the trapping of non-equilibrium structures. Iron-metal hybrids and mutant Hbs will be used in combination with these techniques to test the following five interrelated hypotheses: 1) The two state model must be expanded to encompass the concept of R and T state families. 2) Conformational plasticity modulates allosteric behavior. 3) Hb can act as a combinatorial switch arising in part from intra-dimer communication within the T state. 4) Proposed communication pathways and stereochemical models that account for deviations from the two state model can be tested. 5) Conformational tuning of proximal strain at the heme modulates ligand binding.